Myocardial infarction (MI) is a major health problem worldwide due to its acute nature and lack of effective prevention schemes. Gender difference in ischemic protection exists, with relatively lower MI incidences in pre- menopausal females than age-matched males. Emerging evidence indicates that the female-specific advantage in ischemic protection is mediated by estrogen. In the ischemic protection network, KATP channels (KATP) are postulated to play protective roles, but their relative importance remains to be controversial. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, KATP activity is recorded in cardiac sarcolemmal or mitochondrial inner membrane. Our recent data show that disrupting the SUR2 gene at an earlier exon 3 causes an early lethality and the mutants only lived 8 days. However, disrupting SUR2 at middle exons 12-16 interrupts the SUR2 long forms, but the novel SUR2 short forms remain expressed. We have identified 2 splice variants that are generated by a rare intra-exonic splicing (IES) event in SUR2 mRNA to produce transcripts encoding the 55-kDa SUR2 short forms in heart mitochondria. Characterization of SUR2 KO has revealed an inverse pattern of gender difference in cardioprotection. Completed tests in KO males show that they are constitutively protected, with reduced infarcts after ischemia, while KO females have larger infarcts and cannot be preconditioned. mRNA levels of both IES variants markedly increase in the preconditioned KO males but they reduce dramatically in the preconditioned KO females. This interesting discrepancy offers a new platform of using SUR2 mutant mice to investigate gender difference in ischemic protection. The proposed research intends to explore the molecular mechanisms underlying gender difference in cardioprotection in relation to KATP channels, especially mitochondrial KATP. We hypothesized that estrogen modulates expression of sarcolemmal and mitochondrial SUR2 forms in mice. We further hypothesized that levels of the IES variants encoding the mitochondrial SUR2 short forms are critical to protection. In Aim 1, we will characterize ischemic protection in both genders of WT and KO mice, and study whether estrogen modulates expression of the SUR2 forms. In Aim 2, estrogen regulation in mitochondrial SUR2 will be investigated, and a 55A "rescued" female mouse model will be tested whether we can improve protection. Interactions of estrogen receptor 2 and the IES variants will be explored. Results from this research not only provide new insights in gender-specific response to cardioprotection but also identify new drug targets for future clinical treatments against MI. PUBLIC HEALTH RELEVANCE: The proposed research will employ KATP channel mutant mice that are defective in the sulfonylurea receptor 2 (SUR2) to evaluate gender difference in ischemic protection, regulation of estrogen in sarcolemmal and mitochondrial SUR2 forms and obtain new insights in ion channel regulation in cardiovascular diseases.